chapter12 mechanisms of transcription 胡红霞 04 级生物科学 胡红霞 04 级生物科学...
TRANSCRIPT
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Chapter12Chapter12Mechanisms of Mechanisms of TranscriptionTranscription
胡红霞 胡红霞 0404 级生物科学级生物科学
200431060178200431060178
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The Central Dogma: transcription translation
DNA RNA PROTEIN
Transcription is the first step of the expression of the genome !
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mRNA
transcript
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Similarities vs Differences Transcription && Replication
Transcriptionand replication:1. A new chain
synthesized upon a DNA template
2. In a 5’ to 3’ direction
Transcription :1. Ribonucleotides2. Use RNA polymerases3. Needs no primer (de novo)4. Does not remain base-paired to
the template all the time 5. Less accurate (10-5 vs 10-8)6. Selectively copies certain parts of
the genome and makes anything from one to several hundred , or even thousand, copies of any given section (Recall replication)
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Outlines
1. RNA Polymerases and the Transcription Cycle
2. The Transcription Cycle in Bacteria3. Transcription in Eukaryotes All these topics are very
important !!!
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RNA polymerases
and the transcription cycle
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§1.1 RNA Polymerases
RNA Polymerases Come in Different Forms , but Share Many Features RNA Polymerase performs essentially
the same reaction in all cells, so RNA polymerases from bacteria to humans are highly conserved, especially in those parts of the enzyme directly involved with catalyzing the synthesis of RNA.
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The Subunits of RNA Polymerases
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Eukaryotic cells have three RNA polymerases
• RNA Pol I : transcribes the large ribosomal RNA precursor gene
• RNA Pol II : most studied, transcribes most genes – essentially all protein-encoding genes ( focus)
• RNA Pol III : transcribes tRNA genes, some small nuclear RNA genes and the 5S rRNA gene
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Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerase
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The bacterial RNA polymerase
The core enzyme alone can synthesize RNA
The massive RNA holoenzyme contains 6 subunits: the б subunit, β’ subunit, β subunit , ω subunit, and two α dimer subunits.
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crab claw
the two pincers of the crab claw -- β’ and β subunits
active center cleft (can bind two Mg2+)
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RNA polymerase RNA polymerase holoenzymeholoenzyme
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§1.2 The transcription cycle
RNA polymerases proceeds through a series of well-defined steps :
• Initiation• Elongation• Termination
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Initiation
Important points :A promoter is the DNA sequence that initially
binds the RNA polymerases.The promoter- polymerase complex undergoes
structural changes required for initiation to proceed.
The new ribonucleotide is added to the 3’ end of the growing chain, so transcription always occurs in a 5’ to 3’ direction.
The choice of promoter is the main regulation.
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• Closed complex : form when initially bind to a promoter• Open complex : the DNA strands separate and the
transcription bubble forms• Stable ternary complex : form after an enzyme gets further than
10 bp (that is when the enzyme has escaped the promoter)
Transcription initiation involves three defined steps :
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The phases of the transcription cycle
Initiation
Elongation
Termination
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Elongation
The functions of RNA polymerase during this elongation phase :
1. The catalysis of RNA synthesis;2. Unwinds the DNA in front;3. Re-anneals the DNA behind;4. Dissociates the growing RNA chain
from the template;5. Moves along the DNA template;6. RNA proofreading.
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Transcription Cycle
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Termination
Stops and releases the RNA product once the polymerase has transcribed the length of the gene (or genes) .
In some cells there are specific, well-characterized sequences that trigger it.
In others it is less clear what instructs the termination.
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The Transcription Cycle in Bacteria
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б factor
The RNA polymerase initiate transcription at any point on a DNA molecule; yet the б factor converts core enzyme into the form that initiates only at promoters.
(б70 is predominant in E.coli ) RNA polymerase holoenzyme = RNA polymerase + б factor
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The б subunit is composed of α helices connected by turns and loops.
These elements organize into four domains : N-terminal domain 1 N-terminal domain 2 Linker domain C-terminal domain
After synthesis of a 9-12 nucleotide RNA, the б subunit dissociates from the core polymerase, and the core begins the elongation of the RNA transcript.
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Bacteria Promoters
Vary in strength and sequence , but have certain defining features.
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Characteristic structure of promoters recognized by polymerase containing б70 :
Two conserved sequences: each of 6 nucleotides, centered at about
10 and 35 base pairs upstream of the site where RNA synthesis starts. -10 and -35 regions, or elements;
A nonspecific stretch of 17-19 nucleotides between.
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The б factor mediates binding of polymerase to the promoter
The regions that recognize the -10 and -35 elements of the promoter are region 2 and 4, respectively. ( helix-turn-helix )б region 2 recognizes -10 element
б region 3 recognizes the extended -10 elementб region 4 recognizes -35 element
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Regions of б
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UP-element
An additional DNA elements that binds RNA polymerase and increases binding by providing an additional specific interaction between the enzyme and the DNA.
Is recognized by αCTD of the polymerase
б and α subunits recruit RNA polymerase core enzyme to the promoter
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“Extended –10” element
Another class of б70-promoters lacks a –35 region and has an “extended –10” element. And this element compensates for the absence of a -35 region.
This element is recognized by an α helix in б region 3 through two specific base pairs.
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Transition to the open complex involves structural changesstructural changes in RNA polymerase and in the promoter DNA
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Closed complex Open complex Structural changes:• DNA around the transcription start site is unwound,
forming a bubble of single-stranded DNA• The enzyme also changes (this “melting” occurs between positions -11 and +3) Occurs spontaneously; not require ATP to provide energy.
isomerization
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channels into and out of the open complex
The five channels:
NTP-uptake
RNA-exit
Downstream
DNANontemplate-strand (NT)
Template-Strand (T)
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Upon isomerization there are two striking structural changes in the polymerase1. The pincers at the front clamp down
tightly on the downstream DNA.2. There is a major shift occurs in the N-
terminal region of (region 1.1) .In the closed complex, region 1.1 lies within the active center while in the open complex, it shifts to the outside of the enzyme, allowing the DNA access to the cleft
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Transcription is initiated without a primer
• RNA polymerase can initiate a new RNA chain on a DNA template.
Difficulty : RNA polymerase starts most transcripts with an A, but A-T pair has only two hydrogen bonds.
Various parts of polymerase holoenzyme , including part of б provide specific interactions with the initiating ribonucleotide.
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RNA polymerase synthesizes several short RNAs before entering the elongation phase
• Abortive initiation: the enzyme synthesizes short RNA molecules
less than 10 nucleotides and then releases the transcript.
• Promoter escape :Once the polymerase manages to make an
RNA longer than 10bp, a stable ternary complex containing the enzyme , the DNA template and a growing chain is formed and the elongation starts.
Elongation
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The elongation polymerase is a processive machine that synthesizes and proofreads RNA
Processes DNA: • Enter between the
pincers
• The strands separate
• Reform a double helix behind
RNA polymerase:•Adds new ribonucleotides
•Releases the RNA product
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Attentions :Only 8 – 9 nucleotides of the growing
RNA chain remain base-paired to the DNA template at any given time;
The remainder of the RNA chain is peeled off and directed out of the enzyme through the RNA exit channel
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Proofreading mechanisms
• The enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi, using its active site.
Pyrophosphorolytic editing
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Proofreading mechanisms
Hydrolytic editing• The enzyme
backtracks by one or more nucleotides and cleaves the error-containing sequence. It is stimulated by Gre factor.
Termination
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Transcription is Transcription is terminated by signals terminated by signals within the RNA within the RNA sequencesequence
• Terminators : sequences that trigger the elongating polymerase to dissociate from the DNA and release the RNA chain it has made.
• Two types: Rho-independent terminators Rho-dependent terminators
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Rho-independent terminators
Sequence of a rho-independent terminator:
1.A short inverted repeat (20 bp)
2.A stretch of about 8 A:T base pairs
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Rho-independent terminators
Require A:U base pairs for they are the weakest of all base pairs, then the RNA will more readily dissociate from the template.
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Transcription termination
Polymerase transcribes an inverted repeat ;
Form a stem loop within RNA ;
Cause termination by disrupting the elongation complex .
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Rho-dependent
terminators
Require the action of Rho factor.
Rho ()A ring-shaped protein
with six identical subunits;
Binds to single-stranded RNA as it exits;
Has an ATPase activity The Rho transcription terminator
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Rho is directed to a particular RNA molecule
Rho Binding Specificity :• In the binding sites which consist
of stretches of about 40 nucleotides that remain largely single-stranded and are rich in C residues;
• Only binds those transcripts still being transcribed beyond the end of a gene or operon.
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Transcription
in Eukaryotes
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• Transcription in eukaryotes is undertaken by polymerases closely related to RNA polymerases found in bacteria.
• However, there are some differences between the two cases.
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Differences
1. Eukaryotes have Pol I, II and III , whereas bacteria has only one;
• Eukaryotes require several initiation factors, whereas bacteria require only one --- factor;
• Isomerization to the open complex in eukaryotes require ATP hydrolysis, whereas in bacteria it occurs spontaneously;
• In eukaryotes promoter escape is regulated by the phosphorylation state if the CTD tail;
• Elongation factors and proofreading mechanism;• Termination (some of these will be discussed later)
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RNA polymerase II core promoters RNA polymerase II core promoters are made up of combinations of are made up of combinations of four four different sequence elements different sequence elements
The eukaryotic core promoter : the minimal set of sequence elements required for accurate transcription initiation by Pol IIII
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Regulatory sequence• Beyond –-typically upstream of –- the core
promoter and required for efficient transcription in vivo.
• Categories:1. Promoter proximal elements 2. Upstream activator sequences, UASs 3. Enhancers 4. Silencers 5. Boundary elements 6. Insulators • All these DNA elements bind regulatory
proteins (activators and repressors)
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RNA Pol II forms a pre-RNA Pol II forms a pre-initiation complex with initiation complex with GTFs at the promoterGTFs at the promoter
Pre-initiation complex Pre-initiation complex = GTFs + polymerase+ promoter= GTFs + polymerase+ promoterFunctionsFunctions of the general transcription of the general transcription
factors (GTFs): help polymerasefactors (GTFs): help polymerase1.1. Bind to the promoter ;Bind to the promoter ;2.2. Melt the DNA ;Melt the DNA ;3.3. Escape from the promoter ;Escape from the promoter ;4.4. Begin the elongation phase.Begin the elongation phase.
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TFIID =TBP + IID =TBP + TAFsTAFs
Resulting
Transcription initiation by RNA polymerase IIII
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Transcription initiation by RNA polymerase IIII
The C-terminal domain (CTD) contains repeats of heptapeptide and its phosphorylation helps the promoter escape
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TBP binds to and TBP binds to and distorts DNA using distorts DNA using aa sheet sheet inserted inserted into the minor into the minor groovegroove
• Unusual compared with a helices using by most proteins.
• TBP relies on the ability of TATA sequence to undergo the distortion.
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The other GTFs also The other GTFs also have specific roles in have specific roles in initiationinitiation
• TAFs (TBP associated factors)About 10 TAFs are associated with TBP. Two bind
DNA elements at the promoter; Several bind DNA in the way like histone proteins; Another regulates the binding of TBP to DNA .
• TFIIB A single polypeptide chain, enters the pre-initiation complex after TBP. TFIIB binds to the TBP-TATA complex
asymmetrically.Thus the transcription has an fixed orientation.
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• TFIIF Two-subunit factor; Binding of Pol II–
TFIIF stabilizes the DNA-TBP-TFIIF complex; Required for others.
• TFIIE Two subunits; Has roles in
recruitment and regulation of TFIIH
The other GTFs also have specific roles in The other GTFs also have specific roles in initiationinitiation
TFIIF && TFIIE
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• Nine subunits –- the largest and most complex of the GTFs.
• Function : Controls the ATP-dependent transition of
the pre-initiation complex to the open pre-initiation complex to the open complex;complex;
Two have ATPase activity Two have ATPase activity Another is a protein kinase( promoter Another is a protein kinase( promoter
melting and escape)melting and escape)
The other GTFs also have specific roles in initiationThe other GTFs also have specific roles in initiation
TFIIH
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In vivo, In vivo, transcription transcription initiation requires initiation requires additional proteinsadditional proteins
• Reason for requirement: DNA is packaged into chromatin, not linear
• Different activators interact with different Mediator subunits to bring polymerase to different genes.
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Additional proteins
Assembly of the pre-initiation complex in presence of Mediator, nucleosome modifiers and remodelers, and transcriptional activators
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Mediator consists of many subunits , some conserved from Yeast to Human
Comparison of the yeast and human Mediators
Elongation
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A new set of factors stimulate Pol II elongation and RNA proofreading
• Another set of factors replaced the initiation factors during the transition from initiation to elongation.
• These enzymes are recruited to the phosphorylated CTD .
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Recruitment of enzymes
RNA processing enzymes are recruited by the tail of polymerase
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Various factors stimulating elongation
activates stimulates hSPT5 stimulates • P- TEFb Elongation by Pol II
TAT-SF1
•TFIIS
RNAase Proofreading
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Elongation polymerase Elongation polymerase is associated with a is associated with a new set of protein new set of protein factors required for factors required for various types of RNA various types of RNA processingprocessing
The processing events:• Capping of the 5’ end of the RNA • Splicing (discuss later)• Polyadenylation of the 3’ end of the RNA
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• There is an overlap in proteins involved in elongation and those required for RNA processing.
• Elongation ,termination and RNA processing are interconnected to ensure their proper coordination
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Capping
Methylated guanine
5’-5’ linkage
The first step: the phosphate at the 5’ end of the RNA is removed by RNA triphosphatase
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Capping
The second step: the enzyme guanylyl transferase catalyzes the nucleophilic attack
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Capping
The third step:
Addition of methyl groups by methyl transferase.
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Functions of 5’ cap
• Protection from degradation• Increased translational efficiency• Transport to cytoplasm• Recruitment of the splicing machinery• Continues the transcription (possible)
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Polyadenylation
• Linked with the termination • The polymerase CTD tail is
involved in recruiting the enzymes necessary for polyadenylation as well as with capping and splicing
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Factors involved in Polyadenylation
• The extended C-terminal domain of the largest subunit of RNA (CTD)
• Cleavage and polyadenylation specificity factor (CPSF)
• Cleavage stimulation factor (CstF)• Additional cleavage factors• Poly-A polymerase (PAP)• Poly-A-binding proteins
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Polyadenylation
Transcription of poly-A signal Transcription of poly-A signal
sequencesequence
Cleavage of the RNA
Polyadenylation by poly-A polymerase
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Termination
• RNA polymerase does not terminate immediately when the RNA is cleaved and polyadenylated . Rather, it continues to move along the template, generating a second RNA molecule that can be as long as several hundred nucleotides before terminating.
• It is clear that the polyadenylation signal is required for termination.
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Models of termination
Spontaneous termination
Model 1Transfer of 3’ processing enzymes from the polymerase CTD tail to the RNA
triggers
A conformational change in the polymerase
Reduces processivity of the enzyme
Leading to
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Transfer of the CPSF and CstF from CTD tail to RNA triggers conformational change, reducing processivity
Model Model 11
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Models of termination
• Model 2The absence of a 5’ cap (for the
absence of the capping enzymes on the CTD) on the second RNA molecule is sensed by the polymerase which then recognizes the transcript as improper and terminates.
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Model 2
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RNA Polymerase I and III RNA Polymerase I and III recognize distinct promoters , recognize distinct promoters , using distinct sets of transcription using distinct sets of transcription factors, but still require TBPfactors, but still require TBP
• Two other polymerase in eukaryotes – Pol I and Pol III are related to Pol II, but initiates transcription from distinct promoters and transcribes distinct genes
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RNA polymerase I promoter region
Promoter = the core element + the UCE
Factors needed: Pol I + SL1+ UBF
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RNA Pol III core promoter
Unusual feature of being located downstream
Factors needed: Pol III + TFIIIB + + TFIIIC(tRNA) or TFIIIA(5s rRNA)
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That’s all for mechanisms of transcription !